Haptens and tracers for immunoassays for propoxyphene

ABSTRACT

Disclosed is a substantially optically pure hapten, useful in an immunoassay for dextropropoxyphene and/or nordextropropoxyphene. The hapten corresponds to a specified structural formula (IX). 
     Also disclosed is an immunogen derived from the hapten as well as an antibody raised in response to an immunogen derived from the hapten. 
     Also disclosed is a fluorescent tracer derived from a substantially optically pure compound corresponding to the hapten, the tracer being useful in an immunoassay for dextropropoxyphene and/or nordextropropoxyphene. 
     Also disclosed is an improved immunoassay for determining dextropropoxyphene and/or nordextropropoxyphene in a biological sample involving a step of contacting the sample with antibodies raised in response to the immunogen. Also disclosed is a fluorescence polarization immunoassay (FPIA) for determining dextropropoxyphene and/or nordextropropoxyphene involving a step of contacting the sample with antibodies raised in response to the immunogen, and/or involving a step of contacting the sample with a fluorescent tracer.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention is directed to reagents and methods for performingan immunoassay, particularly a fluorescence polarization immunoassay(FPIA), to determine the presence and/or amount of dextropropoxypheneand/or the principal metabolite of dextropropoxyphene (namely,nordextropropoxyphene) in samples, particularly aqueous, fluidbiological samples such as urine, blood serum or blood plasma, and to animmunoassay based on the reagents. More particularly the invention isdirected to new haptens, immunogens prepared from thehaptens, antibodiesraised against the haptens and immunoassays which utilize reagents andmethods of the invention.

2. Background

Dextropropoxyphene is a narcotic analgesic which has found widetherapeutic use. Unfortunately, however, it has also become a drug ofabuse. Dextropropoxyphene is also known by the following chemical names:[S-(R*,S*)]-alpha-[2-(Dimethylamino)-1-methylethyl]-alpha-phenylbenzeneethanolpropanoate (ester);alpha-d-4-Dimethylamino-3-methyl-1,2-diphenyl-2-butanol propionate;(+)-1,2-Diphenyl-2-propionoxy-3-methyl-4-dimethylaminobutane;(+)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-propionyloxybutane;alpha-d-4-Dimethylamino-3-methyl-1,2-diphenylbutan-2-ol propionate; andd-propoxyphene. Dextropropoxyphene corresponds to the followingstructural formula: ##STR1##

Dextropropoxyphene

As reported by R. J. Flanagan et al in "Measurement ofDextropropoxyphene and Nordextropropoxyphene in Biological Fluids",Human Toxicol. (1984), 3, 103S-114S, various methods have been exploredfor detecting, identifying and measuring the amount of dextropoxypheneand its principal plasma metabolite, nordextropropoxyphene. Examples ofsuch methods include thin-layer chromatography (TLC) and gas-liquidchromatography (GLC) of solvent or solid-phase extracts of urine orgastric contents, as well as a homogeneous enzyme immunoassay method.

U.S. Pat. No. 4,025,501 is directed to compounds for conjugation toantigens for the production of antibodies which recognize(d,1)-propoxyphene and its metabolites in immunoassays. The compounds towhich this patent is directed are prepared by reacting1,2-diphenyl-3-methyl-4-dimethylamino-2-butanol with a dibasic acid toform a half-acid ester, the acid group of which is disclosed as beingactivated for conjugation to amino groups of proteins or polypeptides.

However, presently existing assays for propoxyphene tend to suffer fromdisadvantageously high cross-reactivities for various structurallysimilar compounds. The present invention has a number of objects. Theyinclude, for example, providing new haptens, immunogens prepared fromthe haptens, and antibodies raised in response to the immunogens,suitable for use in immunoassays which are highly discriminating fordextropropoxyphene and its principal metabolite, nordextropropoxyphene,in which immunoassays cross-reactivity for interfering compounds such asmethadone and chlorphenoxamine, among others, is minimized orsubstantially eliminated.

Additionally, presently existing assays for propoxyphene involve the useof racemic mixtures of molecules for preparation of immunogens to raiseantibodies against propoxyphene. However, propoxyphene can exist in fourdifferent isomeric forms because of the presence of two optically activecenters within the molecule. Thus, utilization of such racemic mixturesfor the preparation of immunogens to raise antibodies is believed toresult in the production of at least four types of antibodies of whichonly about 25% will detect the optical form of propoxyphene that isrelevant to the assay. The form of propoxyphene which is available onthe market is an optically active form of the drug. Moreover, of thefour isomeric forms of propoxyphene, there is only one form whichappears to have any substantial efficacy in man, namelydextropropoxyphene. Accordingly, particularly for quantitative assaysfor that form of propoxyphene having efficacy in man, and/or for itsmost important metabolite, it would be desirable to provide antibodies,a larger proportion of which are reactive to the effective form of thedrug than to ineffective forms of the drug.

The present invention is related to immunoassays, particularlycompetitive immunoassays, involving reagents and techniques particularlysuitable for determining the presence and/or amount ofdextropropoxyphene and/or nordextropropoxyphene in biological fluids.The present invention can provide, among others, an advantage ofallowing for an advantageously effective determination of the amount ofdextropropoxyphene and/or its principal metabolite,nordextropropoxyphene, with minimization of interference from otherrelated compounds. The present invention is in part based on new,substantially optically pure haptens, which can be utilized in thepreparation of immunogens and/or tracers suitable for use inimmunoassays.

SUMMARY OF THE INVENTION

The invention provides for a substantially optically pure hapten, usefulin an immunoassay for dextropropoxyphene and/or nordextropropoxyphene.The hapten of the invention corresponds to the structural formula (IX):##STR2## In formula (IX), Z is a --NH-- moiety and is present when m=1.X is a functional group selected from the group consisting of --CHO,--COOH, --NH₂, and --COOR in which R is a C₁ -C₃ alkyl group. In formula(IX), m can equal 0 or 1, and n can equal an integer of from 1 to 3.

The invention also provides for an immunogen derived from a hapten ofthe invention.

The invention also provides for an antibody raised in response to animmunogen derived from a hapten of the invention.

The invention also provides for a fluorescent tracer derived from asubstantially optically pure compound of the invention, the tracer beinguseful in an immunoassay for dextropropoxyphene and/ornordextropropoxyphene. The substantially optically pure compoundcorresponds to the hapten defined in formula (IX).

The invention also provides for an improved immunoassay for determiningthe presence or amount of dextropropoxyphene and/ornordextropropoxyphene in a biological sample. The improved immunoassaycomprises a step of contacting the sample with an antibody (orantibodies) raised in response to an immunogen of the invention.Moreover, the invention provides for a fluorescence polarizationimmunoassay (FPIA) for determining the presence or amount ofdextropropoxyphene and/or nordextropropoxyphene in a biological sample.The FPIA comprises a step of contacting the sample with antibodiesraised in response to an immunogen of the invention, and/or comprises astep of contacting the sample with a fluorescent tracer of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

A hapten of the invention is substantially optically pure, and isparticularly useful in an immunoassay for dextropropoxyphene and/or fornordextropropoxyphene, a metabolite of dextropropoxyphene. As usedherein, the phrase "substantially optically pure" means that the producthapten contains less than or equal to 10 percent, preferably less thanor equal to 5 percent, and most preferably less than or equal to 2percent, by weight of the dextrorotatory (d or +) enantiomer of thehapten based on the sum by weight of the dextrorotatory and levorotatory(1 or -) enantiomers. A hapten of the invention corresponds to theformula (IX): ##STR3## In formula (IX), Z represents a divalent --NH--moiety and is present when m=1. X is a functional group selected fromthe group consisting of --CHO, --COOH, --NH₂, and --COOR in which R is aC₁ -C₃ alkyl group. It is preferred that X be an aldehyde group. It isalso preferred that R be an ethyl group. In formula (IX), m can equal 0or 1, preferably 0, and n can equal an integer of from 1 to 3,preferably 3. The functional group, X, in formula (IX) is a functionalgroup suitable for utilization, for example, in attaching anantigenicity-conferring moiety to the hapten, for example, by reactiondirectly, or via an intermediate step, with a co-reactive functionalgroup from an antigenicity-conferring carrier.

Haptens of the invention can be prepared by the following illustrativegeneral procedures. Typically, the optically active dextropropoxyphenederivatives (haptens) of the invention are prepared by reaction ofdextropropoxyphene hydrochloride with a suitable reducing agent. Theresulting alcohol is then derivatized by treatment with an acyl halideor isocyanate reagent, which contains a second functionality capable ofbeing transformed into another functional group. This new functionalgroup allows the dextropropoxyphene derivative thereby produced to becoupled to antigenic molecules. The second functionality can be an esteror an olefin. The overall methodology used for elaboration of thelinkage of the dextropropoxyphene derivative in this manner is centralto the obtainment of haptenic dextropropoxyphene derivatives of highoptical purity.

Reduding agents which can be used in the preparation of haptens of theinvention include reducing agents such as lithium aluminum hydride,di-isobutylaluminum hydride, bis(methoxyethoxy)aluminum hydride, and thelike, with lithium aluminum hydride being preferred.

The acyl halide or isocyanate reagents described above are preferablyaliphatic acid chlorides or aliphatic isocyanates such as pentenoylchloride, butenoyl chloride, ethyl isocyanatoacetate, ethyl3-isocyanatopropionate and the like, with pentenoyl chloride beingpreferred for hapten formation. Acid chlorides and isocyanates derivedfrom aromatic acids are less preferred.

Modification of the second functionality present in the acid halidemolecule to allow for coupling to an antigenic molecule utilizes twotypes of reagents, depending on the type of functionality present.Unsaturated acid chlorides, such as butenoyl chloride, give rise todextropropoxyphene derivatives containing an olefin. Olefins can betransformed to aldehydes using a variety of oxidizing reagents,including lead tetraacetate, sodium periodate, ozone followed by asuitable reductive workup, and the like. Unsaturated isocyanates alsogive rise to olefinic dextropropoxyphene derivatives which contain acidsupon treatment with suitable cleavage reagents. These reagents includeacid, base, aromatic sulfide salts, silyl iodides, and the like. Acidchlorides containing a second ester functionality also can give rise todextropropoxyphene derivatives containing an acid.

Both the dextropropoxyphenic aldehydes and acids obtained are linked toantigenic molecules by methods well known to those skilled in the art.

It has been found that fluorescence polarization immunoassays performedutilizing antisera raised from immunogens prepared from haptens of theinvention can provide a particularly high specificity fordextropropoxyphene and/or nordextropropoxyphene and an especially lowcross-reactivity for interfering compounds such as methadone andchlorophenoxamine, among others.

An immunogen of the invention is derived from a substantially opticallypure hapten of the invention. An immunogen of the invention isparticularly useful in an immunoassay for dextropropoxyphene and/ornordextropropoxyphene. The immunogen corresponds to the formula (X):##STR4## wherein Z is as defined for the hapten corresponding to formula(IX) above. Y in formula (X) is a divalent moiety for linking for(CH₂)_(n) group to the antigenicity-conferring moiety A. Y can be--NH--, --(CO)NH--, or --NH(CO)-- with --NH-- being preferred. It is tobe understood that wherever in the specification and claims herein, adivalent moiety for linking two other structures together is specified,e.g., --(CO)NH-- as Y for linking (CH₂)_(n) and A, the left hand portionof the divalent moiety is attached to the structure on the left and theright hand portion is attached to the structure on the right ((i.e., forthis example (CH₂)_(n) --(CO)NH--A)). In formula (X), the subscript, m,can be 0 or 1, with 0 being preferred, and the subscript n can be aninteger of from 1 to 3 with 3 being preferred. Theantigenicity-conferring carrier moiety, A, can be selected from a widevariety of antigenicity-conferring carrier moieties. As can beappreciated from formula (X), the moiety A represents the residue of anantigenicity-conferring carrier bound via Y to the haptenic portion ofthe immunogen of formula (X).

Covalent linkage of the haptenic materials described herein toantigenicity-conferring materials can be accomplished by methods wellknown in the art, the choice of which will be dictated by the nature ofthe linking functionality in the dextropropoxyphene derivative (i.e., Xin formula (IX)) and the carrier chosen for the linkage.

Typically, the antigenicity-conferring carrier moiety, A, is provided byreacting the functional group X of a hapten corresponding to formula(IX) with a co-reactive functional group of an antigenicity-conferringcarrier such as, for example, a naturally occurring or syntheticpoly(amino-acid) by generally known preparative techniques. Typically,in preferred embodiments of the invention, the naturally occurringpoly(amino-acid), bovine thyroglobulin (BTG), is utilized as theantigenicity-conferring carrier to provide the moiety, A, in structuralformula (X), but it is to be understood that other protein carriers canbe utilized, including for example, albumins and serum proteins such asglobulins, lipoproteins, ocular lens proteins, and the like. Someillustrative antigenicity-conferring protein carriers include bovineserum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin,bovine gamma globulin (BGG), thyroxine binding globulin, etc.Alternatively, synthetic poly(amino-acids) can be utilized such aspolylysine, etc.

For example, a hapten in which X of formula (IX) is carboxyl, can becoupled to bovine serum albumin, preferably under conditions normallyused to form amide bonds which conditions are well known to thoseskilled in the art, by utilizing as the coupling agent, for example, acarbodiimide, especially a water soluble carbodiimide such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or1-cyclohexyl-3-(2-morpholinoethyl) carbodiimidemetho-p-toluenesulfonate. The same reagents can be used in the casewhere X of the hapten is a --NH₂ group, in which case an amide bond isformed with a carboxyl group on the bovine serum albumin. When Q of thehapten is --CHO, the aldehyde can be reductively aminated to acorresponding amine functional group. Other transformations of thealdehyde into useful haptens are obvious to one skilled in the art.

Antibodies of the present invention are prepared by developing an immuneresponse in animals to immunogens of the invention. The immunogen isadministered to animals such as rabbits, mice, rats, sheep or cows by aseries of injections according to techniques generally known in the art.An antibody, according to the present invention, is raised in responseto an immunogen of the invention which is derived from a substantiallyoptically pure hapten of the invention. Both polyclonal and monoclonalantibodies recognize specific epitopes on an immunogen, and, whiletypically polyclonal antibodies have been utilized in the presentinvention, both may be suitable. Polyclonal antibodies consist of amixture of multiple antibodies, each recognizing a specific epitope,whereas monoclonal antibodies are produced by cells secreting a singleantibody recognizing a specific epitope. Techniques for preparingpolyclonal antibodies generally are well known in the art.

Monoclonal antibodies may be prepared by injecting animals, such as miceor rats, intraperitoneally, subcutaneously, intravenously, or in someother manner, with an antigen, namely an immunogen corresponding toformula (X) above, to elicit an immune response in the animals (namely,the production of antibodies which are specific for the antigen). Serafrom the animals are drawn, and the sera are tested to determine thetiter of antibody in the sera (to determine whether or not the animalelicited the desired immune response, and to what extent). Those animalsin which the desired immune response has been produced are permitted torest for approximately two to three months. After this two-month tothree-month period of time, and approximately three days prior to theanticipated fusion of B-lymphocyte cells (cells which, upon stimulationby antigen, mature into plasma cells which synthesize antibody, andwhich are also referred to as B cells) with myeloma cells (tumor cells),a boost injection of the antigen is administered to these animals.B-lymphocyte cells are then removed from the spleens of these animals bystandard procedures, and the B-lymphocyte cells are then fused withmyeloma fusion partners according to standard procedures, such as thosedescribed in Kohler and Milstein, "Continuous Culture of Fused CellsSecreting Antibody of Predefined Specificity," Nature, 256, 495 (1975).The B-lymphocyte-myeloma fusions are then plated in multiwell tissueculture plates containing HAT media, or other suitable media. Theresulting cultures are fed with HT media, or other suitable media, andfetal bovine serum or calf bovine serum on or about the fifth andseventh days after the fusion of the cells and then tested on or aboutthe tenth day after the fusion for the presence of antibody which isspecific for the antigen. Specific desirable hybrids are then cloned bylimiting dilution. (Hybrid cells are diluted in differing amounts of HTmedia, or other suitable media, and plated out in tissue culture platesin order to isolate a single desired clone.) Established clones are thenretested for specificity to a broader panel of cross reactants.

The amount of the resulting monoclonal antibodies produced by a desiredclone can then be scaled up to produce a sufficient quantity of antibodyfor purification in either: (1) tissue culture (by expanding the numberof cells in tissue culture, or HT media); or (2) mice for ascites. Themonoclonal antibodies can be scaled up in mice by injecting hybrid cellsinto the abdominal cavity of mice and allowing the cells to grow(usually for about 7 days). The ascites is harvested from the mice bysacrificing the mice, collecting the ascites fluid, and purifying theascites fluid. BALB/c mice are the most common strain of laboratorymouse used for this process, and they can be obtained from any mousevendor. Pristane, should be injected into the mice to stimulate theirimmune systems to produce B and T cells (about two or three weeks beforethe hybrid cells are injected into the mice) which serve as a feederlayer for the clone cells that are injected into the mice. This isperformed to provide a suitable environment in which the hybrid cellscan grow.

The invention also provides for improved immunoassays for determiningthe presence or amount of dextropropoxyphene and/ornordextropropoxyphene in biological samples. An improved immunoassay ofthe invention includes (comprises) a step of contacting the sample to bedetermined with antibodies raised in response to an immunogen of theinvention. It is contemplated that any immunoassays for propoxypheneand/or nordextropropoxyphene utilizing haptens, immunogens, and/orantibodies raised against immunogens, according to the invention, arewithin the scope of the present invention. Examples of immunoassaysinclude radioimmunoassays (RIAs), enzyme immunoassay (EIAs), enzymelinked immunosorbent assays (ELISAs) and fluorescent polarizationimmunoassays (FPIAs). In a fluorescent polarization immunoassay (FPIA),a fluorescent tracer of the invention may be utilized either with orwithout utilization of antibodies raised in response to an immunogen ofthe invention.

A fluorescent tracer of the invention can be thought of as being derivedfrom a substantially optically pure compound corresponding to a haptenof the invention. A tracer of the invention is useful in an immunoassayfor dextropropoxyphene and/or nordextropropoxyphene and corresponds tothe formula (XI): ##STR5## wherein Z is as defined for the haptencorresponding to formula (IX) above with subscript m equal to 0 or 1,preferably 1. In formula (XI), Y is a divalent radical selected from--NH--, --(CO)NH--, and --NH(CO)-- with --(CO)NH-- being preferred. Thesubscript n is an integer of from 1 to 3 and is equal to 1 in apreferred tracer of the invention. In formula (XI), FL is afluorescence-conferring moiety, and Y serves to link thefluorescence-conferring moiety, FL, to the divalent radical --(CH₂)_(n)--. The fluorescence-conferring moiety, FL, can be selected from avariety of fluorescence-conferring moieties.

As can be appreciated from formula (XI), the moiety FL represents themonovalent residue of a fluorescence-conferring compound bound via Y tothe remainder of the tracer. In preferred embodiments of the invention,the fluorescence-conferring moiety of the fluorescent tracer is amonovalent residue of fluorescein or a monovalent residue of afluorescein derivative. By way of example, any of the followingfluorescein derivatives can be utilized: FL--NH₂, fluorescein amine;FL--CH₂ NH₂, aminomethylfluorescein; and FL--COOH, carboxyfluorescein.As used herein, FL stands for a fluorescein moiety corresponding to thefollowing formula (XII): ##STR6## In a preferred embodiment of theinvention, FL--NH₂ is utilized for preparation of the tracer, preferablywherein the --NH2 group is bonded to FL at either the number 5- or6-position (see FIG. XII above), typically at the 5-position.

Tracers of the invention generally can be prepared by linking anappropriate fluorescent compound to a hapten of the inventionrepresented by formula (IX) above in which X represents a functionalgroup suitable for utilization in attaching the fluorescent compound tothe hapten, for example, by reaction directly, or via an intermediatestep, with a co-reactive functional group from the fluorescent compound.Examples of functional groups for the hapten include: --COOH, --NH₂, and--COOR in which R is a C₁ -C₃ alkyl group. Reaction conditions forreacting such functional groups of the hapten as represented by X withco-reactive functional groups of a fluorescent compound are well knownin the art.

Normally, competitive binding immunoassays are utilized according to themethod of the invention to determine the presence and/or amount ofdextropropoxyphene and/or nordextropropoxyphene in a biological sample.Typically, competitive binding immunoassays are used for measuringligands in a test sample. For purposes of this disclosure, a "ligand" isa substance of biological interest (dextropropoxyphene and/ornordextropropoxyphene) to be quantitatively determined by a competitivebinding immunoassay technique. The ligand competes with a labeledreagent (a "ligand analog" or "tracer") for a limited number of ligandbinding sites on antibodies specific to the ligand and ligand analog(herein, antibodies prepared in response to an immunogen of theinvention). The concentration of ligand in the sample determines theamount of ligand analog which binds to the antibody, and the amount ofligand that will bind to the antibody is inversely proportional to theconcentration of ligand in the sample, because the ligand and the ligandanalog each bind to the antibody in proportion to their respectiveconcentrations.

In one embodiment of the invention, fluorescence polarizationimmunoassay (FPIA) techniques are utilized for determining the amount oftracer-antibody conjugate produced in a competitive binding immunoassay.Such procedures are based on the principle that a fluorescent labeledcompound, when excited by plane polarized light, will emit fluorescencehaving a degree of polarization inversely related to its rate ofrotation. Accordingly, when a tracer-antibody conjugate having afluorescent label is excited with plane polarized light, the emittedlight remains highly polarized because the fluorophore is constrainedfrom rotating between the time that light is absorbed and emitted. Incontrast, when an unbound tracer is excited by plane polarized light,its rotation is much faster than the corresponding tracer-antibodyconjugate and the molecules become more randomly oriented. As a result,the light emitted from the unbound tracer molecules is depolarized.

More specifically, a preferred FPIA method of the present invention fordetermining the presence or amount of dextropropoxyphene and/ornordextropropoxyphene in a sample comprises the steps of: (a) contactinga sample with: (1) an antiserum containing monoclonal or polyclonal,typically polyclonal, antibodies which have been raised in response toan immunogen of the invention; and (2) a fluorescent tracer of theinvention, the fluorescent tracer being capable of producing adetectable fluorescence polarization response to the presence of theantiserum; (b) passing plane polarized light through the resultingsolution from step (a) to obtain a fluorescence polarization response;and (c) detecting the fluorescence polarization response of the solutionof step (b) as a measure of the presence or amount of dextropropoxypheneand/or nordextropropoxyphene in the sample.

By maintaining constant the concentration of fluorescent tracer andantibody, the ratio of dextropropoxyphene and/ornordextroproxyphene-antibody complex to fluorescent tracer-antibodycomplex that is formed is directly proportional to the amount ofdextropropoxyphene and/or nordextropropoxyphene in the sample. Uponexciting the mixture with linearly polarized light and measuring thepolarization (in units of millipolarization) of the fluorescence emittedby a fluorescent tracer and a fluorescent tracer-antibody complex, oneis able to quantitatively determine the amount or qualitativelydetermine the presence of dextropropoxyphene and/ornordextropropoxyphene in the sample.

The results can be quantified in terms of net millipolarization unitsand span (in millipolarization units). The measurement of netmillipolarization units indicates the maximum polarization when amaximum amount of the fluorescent tracer is bound to the antibody, inthe absence of any dextropropoxyphene and/or nordextropropoxyphene. Thehigher the net millipolarization units, the better the binding of thetracer to the antibody. The assay span is the difference between the netmillipolarization values obtained when the maximum amount of tracer isbound in the absence of any dextropropoxyphene and/ornordextropropoxyphene and the net millipolarization obtained when aspecific amount of dextropropoxyphene and/or nordextropropoxyphene ispresent in the sample. A larger span allows for more millipolarizationunits to be placed between each of the calibrators of the standard curvegenerated for the assay, thereby providing better assay precision which,in turn, results in a better numerical analysis of the data obtained. Itis important to note that the span varies depending on the sample sizeused which, in turn, may alter the preferred combination.

Fluorescent tracers of the present invention are substantially opticallypure. These tracers have the particular advantage in instances whereantisera based on polyclonal antibodies are utilized. Becausedextropropoxyphene is essentially optically pure in the body, the use oftracers which are substantially optically pure in combination withantibodies derived from substantially optically pure immunogens havebeen found to allow for an enhanced signal across the chosen dynamicrange in a dextropropoxyphene and/or nordextropropoxyphene assayutilizing FPIA techniques. One resultant advantage is that FPIA assaysof the present invention can achieve sensitivities of the order of 20.0nanograms/milliliter (ng/ml) of dextropropoxyphene and/ornordextropropoxyphene in the sample.

Some significant features of the most preferred combination offluorescent tracer of the present invention and immunogen of the presentinvention include: (1) the high degree of specificity of the antibodies,generated in response to the immunogen, for dextropropoxyphene and/ornordextropropoxyphene, and (2) minimal cross reactivity of theseantibodies to potential interferants.

The pH at which an FPIA method of the present invention is practicedmust be sufficient to allow the fluorescein moiety of the fluorescenttracer to exist in its open form. The pH may range from about 3 to 12,more usually in the range of from about 5 to 10, most preferably fromabout 6 to 9. Various buffers may be used to achieve and maintain the pHduring the FPIA procedure. Representative buffers include borate,phosphate, carbonate, tris, barbital, citrate and the like. Theparticular buffer employed is not critical to the present invention, butthe tris, phosphate and citrate buffers are preferred. The cationportion of the buffer will generally determine the cation portion of thetracer salt in solution.

Riboflavin binding protein (RBP) is added to the sample or to one ormore of the assay reagents to bind any riboflavin present in the sampleinto RBP-riboflavin complexes, thus eliminating potential fluorescenceinterference. RBP is a protein of approximately 32,000 M.W. which isisolated from egg whites. Upon isolation from the egg, each molecule ofRBP contains one molecule of riboflavin. This, the holoprotein form ofRBP, must be converted to the apoprotein form by dialysis, under acidicconditions, to remove the bound riboflavin. The RBP apoprotein utilizedin the present invention is commercially available from Sigma ChemicalCompany, St. Louis, Mo. The amount used is not critical, provided asufficient quantity is used to bind all free riboflavin in the sample.

A fluorescent polarization immunoassay of the present invention is a"homogeneous assay," which means that the end polarization readings aretaken from a solution in which bound tracer is not separated fromunbound tracer. This is a distinct advantage over heterogeneousimmunoassay procedures, such as those where the bound tracer must beseparated from the unbound tracer before a reading can be taken.

The reagents for the fluorescence polarization assay of the presentinvention comprise: (1) polyclonal or monoclonal, typically polyclonal,antibodies, for dextropropoxyphene; and (2) fluorescent tracer reagent.

Additionally, largely conventional solutions including a pretreatmentsolution, a dilution buffer, dextropropoxyphene calibrators anddextropropoxyphene controls are desirably prepared. Typical solutions ofthese reagents, some of which are described below, are commerciallyavailable in assay "kits" from Abbott Laboratories, Abbott Park, Ill.

All percentages expressed herein are weight/volume unless otherwiseindicated. The preferred reagents, calibrators and controls for apreferred fluorescence polarization immunoassay of the present inventioncan be found in Example 7 infra.

The preferred FPIA procedure is especially designed to be used inconjunction with the Abbott TD_(X)® Clinical Analyzer, the Abbott TD_(X)FL_(X) ™ or the Abbott AD_(X)® Drugs of Abuse System, all three of whichare available from Abbott Laboratories, Abbott Park, Ill. Thecalibrators, controls, or unknown samples are pipetted directly into thesample well of the TD_(X)® sample cartridge. One of the advantages ofthis procedure is that the sample does not require any specialpreparation. The assay procedure from this point is fully automated.

If a manual assay is being performed, the sample is mixed with thepretreatment solution in dilution buffer and a background reading istaken. The fluorescence tracer is then mixed with the assay. Theantibody is then finally mixed into the test solution. After incubation,a fluorescence polarization reading is taken.

The fluorescence polarization value of each calibrator, control orsample is determined and is printed on the output tape of an instrument,such as the Abbott TD_(X)® Analyzer, TD_(X) FL_(X) ™ or AD_(X)® System.A standard curve is generated int he instrument by plotting thepolarization of each calibrator versus its concentration using anonlinear regression analysis. The concentration of each control orsample is read off of the stored calibration curve and printed on theoutput tape.

With respect to the foregoing preferred procedure, it should be notedthat the tracer, antibody, pretreatment solution, wash solution,calibrators and controls should be stored between about 2 degrees C. andabout 8 degrees C. while the dilution buffer should be stored at ambienttemperature. A standard curve and controls should be run every twoweeks, with each calibrator and control run in duplicate. All samplescan be run in duplicate.

The following examples are provided to further illustrate embodiments ofthe invention and should not be construed as a limitation on the scopeof the invention.

The following general experimental procedures were utilized in thepreparation of the haptens of the following examples.

EXAMPLE 1

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-butan-2-ol, correspondingto formula (I) below, for use in the preparation of haptens and tracersof the invention. ##STR7##

To a cooled (0° C.) suspension of 200 milligrams (mg, 0.53 mmol) ofdextropropoxyphene hydrochloride in 3.0 milliliters (ml) of anhydroustetrahydrofuran (THF) was added 22 mg (0.59 mmol) of lithium aluminumhydride. The mixture was warmed to ambient temperature and stirred for 4hours (hr). The reaction was then quenched with water, basified with 2molar (M) aqueous potassium hydroxide solution, and extracted 3 timeswith 10 ml aliquots of ethyl acetate. The combined extracts were driedover MgSO₄, filtered, and concentrated to 164 mg of a clear colorlessoil of (2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-butan-2-ol, whichwas used subsequently without further purification.

EXAMPLE 2

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-ethoxycarbonylmethylaminocarbonyloxy)-butane,corresponding to formula (II) below. ##STR8##

A solution of 127 mg (0.45 mmol) of the clear colorless oil (of thealcohol) prepared immediately above in a mixture of 1.0 ml benzene and1.0 ml toluene was fractionally distilled, and only 1.0 ml of distillatewas collected. The residual solution was cooled, and 0.15 ml (1.34 mmol)of ethyl isocyanatoacetate, OCN--CH₃ COOCH₂ CH₃, was injected. Theresulting solution was refluxed for 1 hr, cooled and quenched with 2drops of water. The resulting mixture was concentrated on a rotaryevaporator and chromatographed directly on a 1×18 centimeter (cm) columnof silica gel, packed with chloroform and eluted with 50 ml aliquots of2%, 6%, and 10% by volume solutions, respectively, of methanol inchloroform. A total of 269 mg of a clear colorless oil of the ester(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-(ethoxycarbonylmethylaminocarbonyloxy)-butanewas obtained.

EXAMPLE 3

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-(carboxymethylaminocarbonyloxy)-butane,corresponding to formula (III) below, a hapten of the invention.##STR9##

Potassium hydroxide (24 mg, 0.43 mmol) was added to a solution of 118 mg(0.29 mmol) of the clear colorless oil of the ester (prepared in Example2 above) in a mixture of 0.75 ml tetrahydrofuran and 0.75 ml water. Thesolution was stirred for 12 hr at ambient temperature, acidified to pH7, and concentrated. The concentrate was redissolved in methanol and thesolution streaked onto two 0.50 millimeter (mm)×20 cm×20 cm plates,which were developed with 30% by volume solution of methanol inchloroform (methanol/chloroform). The product was eluted from thepulverized, scraped band with 80% methanol/chloroform, and concentratedto 50 mg of a white solid of the acid(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-(carboxymethylaminocarbonyloxy)-butane.

EXAMPLE 4

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-(5-fluoresceinylaminocarbonylmethylaminocarbonyloxy)-butane,corresponding to formula (IV) below, a tracer of the invention.##STR10##

To a cooled (0° C.) solution of 3.6 mg (9.3 micromole, μmol) of thewhite solid acid, of Example 3 above, in 100 microliters (μl) ofdimethylformamide, was added 1.2 μl (9.3 μmol) isobutyl chloroformate.The resulting solution was stirred for 1 hr, allowing warming to ambienttemperature, and was then recooled to 0° C. 5-Fluoresceinamine (3.2 mg,9.3 μmol) was then added, and the resulting solution was stirred for 12hr at ambient temperature. The mixture was concentrated, redissolved inmethanol, and streaked onto a 0.25 mm×20 cm×20 cm plate. Developmentwith 20% methanol/chloroform, and elution of the pulverized band with80% methanol/chloroform, provided 8.1 mg of an orange solid. This solidwas rechromatographed on an additional 0.25 mm plate, eluting with70:30:2 chloroform:methanol:glacial acetic acid. The band was pulverizedand eluted with 80% methanol/chloroform to provide 32 mg of an orangesolid. This solid was rechromatographed twice more on additional 0.25 mmplates, developed with 50% methanol/chloroform each time, and eachpulverized band was eluted with 80:20:1 methanol:chloroform:concentratedammonium hydroxide. 4.5 mg of an orange solid of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-(5-fluoresceinylaminocarbonylmethylaminocarbonyloxy)-butane,was obtained after the final purification.

EXAMPLE 5

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-[1-(but-4-enyl)carbonyloxy]-butane, corresponding to formula (V) below, a precursor forpreparation of a hapten of the invention. ##STR11##

A solution of 153 mg (0.54 m mol) of the clear colorless oil (thealcohol) prepared in Example 1 above in a mixture of 1.0 ml benzene and1.0 ml toluene was fractionally distilled, and only 1.0 ml of distillatewas collected. The residual solution was cooled to ambient temperature,and a solution of 1.00 mmol of 4-pentenoyl chloride in benzene wasadded. The resulting mixture was gradually warmed; the benzene wasremoved by distillation; and the residual mixture was refluxed for 2 hr.The precipitate which formed was filtered and washed with 2 ml oftoluene. The combined filtrate and washings were diluted in chloroform,extracted with 2M potassium hydroxide solution dried over MgSO₄,filtered, and concentrated. The resulting olefinic ester was usedwithout further purification.

EXAMPLE 6

This example illustrates the preparation of(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-[1-(propan-3-al)carbonyloxy]-butane,corresponding to formula (VI) below, a hapten of the invention.##STR12##

To a cooled (0°) solution of 40 mg (0.11 mmol) of the olefinic ester,prepared in Example 5 above, in 0.50 ml of dichloromethane, was added 50μl of trifluoroacetic acid. The resulting mixture was stirred for 5minutes after warming to ambient temperature, concentrated, andredissolved in 5.0 ml methanol. The resulting solution was ozonizeduntil a blue color persisted. Excess ozone was removed by passage of astream of argon, and the resulting colorless reaction mixture wasquenched with 0.10 ml of dimethyl sulfide. The reaction mixture waswarmed slowly to ambient temperature (over about 1 hr) and concentratedto provide 69 mg of a light-yellow oil of the hapten,(2S,3R)-4-Dimethylamino-1,2-diphenyl-3-methyl-2-[1-(propan-3-al)carbonyloxy]-butane.The hapten was conjugated to protein to produce an immunogen of theinvention according to the procedure of Example 7 below.

EXAMPLE 7

This example illustrates the preparation of an immunogen of theinvention illustrated in formula (VII) below in which BTG represents abovine thyroglobulin moiety attached through an amide linkage to theremainder of the compound. ##STR13##

(a) An amount of 53.0 mg of the hapten (VI) from example 6 was added to1.0 ml of deionized water and 0.5 ml of dimethylsulfoxide (DMSO). Avolume of 0.750 ml (containing 26.5 mg of hapten) of the resultingsolution was added to 10.4 ml of a solution of bovine thyroglobulin(containing 10 mg/ml of thyroglobulin and 0.05 molar (M) sodiumphosphate and having a pH=7.0) with stirring for 30 minutes at roomtemperature. Five additions of 50 mg each of sodium cyanoborohydridewere added, with 2 hour intervals between additions, to the resultingmixture with the pH of the mixture having been adjusted with 0.1 normal(N) HCl to about 7.0 before each addition. After the last addition ofsodium cyanoborohydride, the mixture was stirred for 2 hours. Next, themixture was dialyzed in a cellulose dialyzing tube against 0.05M sodiumphosphate at pH=7.5 for 36 hours with four changes of dialysate. Afterdialysis, the mixture was centrifuged in Sorvall at 10,000 revolutionsper minute (rpm) for 10 minutes. The supernatant was found to contain9.78 mg/ml of protein via the Lowry protein concentration determiningmethod.

(b) Antisera was produced from the immunogen of part (a) immediatelyabove and was utilized in fluorescence polarization immunoassays (FPIAs)directed to the determination of propoxyphene in urine samples.

The configuration of the reagents, calibrators and controls for theFPIAs is as follows:

1. The tracer formulation is 100 nanomolar tracer in: 0.1 molar sodiumphosphate buffer at pH=6.3, 0.01 percent bovine gamma-globulin, 5percent 5-sulfo-salicylate, and 0.1 percent sodium azide.

2. The antiserum formulation comprises sheep serum diluted with: 0.1molar tris buffer at pH=7.5, 2 percent ethylene glycol, 0.1 percentsodium azide, and 0.05 percent bovine gamma-globulin.

3. The pretreatment solution consists of: 0.1 molar tris buffer atpH=7.5, 0.01 percent bovine gamma-globulin, 0.1 percent sodium azide,and 4 mg/ml riboflavin binding protein.

4. The wash solution consists of: 0.1 molar sodium phosphate buffer atpH=7.5, 0.1 percent sodium azide, and 0.01 percent bovinegamma-globulin.

5. The dilution buffer consists of: 0.1 molar sodium phosphate buffer atpH=7.5, 0.1 percent sodium azide, and 0.01 percent bovinegamma-globulin.

6. The calibrator/control diluent consists of: normal human urine and0.1 percent sodium azide.

7. Propoxyphene calibrators consist of propoxyphene in treated normalhuman urine at concentrations of 0.0, 150.0, 300.0, 500.0, 1000.0, and1500.0 nanograms per milliliter.

8. Propoxyphene controls consist of: propoxyphene in treated normalhuman urine at concentrations of 200.0, 400.0 and 900.0 nanograms permilliliter.

(c) The assays used a six point callibration curve withdextropropoxyphene as the calibrator. The calibration curve has a twoweek minimum stability and a range of 0.0 ng/ml to 1500.0 ng/ml. Theassays have a sensitivity of 20.0 ng/ml. Sensitivity is defined as thelowest measurable concentration which can be distinguished from zerowith 95 percent confidence.

Reproducibility on the TD_(x) ® instrument was determined on tendifferent days over a period of two weeks by assaying five replicateseach of dextropropoxyphene in human urine at 200, 400 and 900 ng/ml. Theconcentration of each was determined from a single standard curve run onthe first day of the study. The results are summarized in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        TDx ® DATA Concentration (ng/ml)                                          ______________________________________                                        Target Value   200        400     900                                         No. Samples = 50                                                              Mean           187.85     388.27  887.33                                      SD Within Run  2.87       6.38    12.88                                       CV Within Run (%)                                                                            1.53       1.64    1.45                                        SD Between Run 9.30       15.70   25.55                                       CV Between Run (%)                                                                           4.95       4.04    2.88                                        SD Total       9.73       16.95   28.61                                       CV Total (%)   5.18       4.37    3.22                                        ______________________________________                                    

Reproducibility on the AD_(x) ® instrument was determined over fifteendifferent runs, in combination, batch and panel modes, by assaying fourreplicates each of dextropropoxyphene in human urine at 200, 400, and900 ng/ml. The concentration of each was determined from a standardcurve run in duplicate on the first day of the study. The results aresummarized in Table 2 below

                  TABLE 2                                                         ______________________________________                                        ADx ® DATA Concentration (ng/ml)                                          ______________________________________                                        Target Value   200        400     900                                         No. Samples = 60                                                              Mean           195        401     906                                         SD Within Run  4.55       10.47   19.42                                       CV Within Run (%)                                                                            2.34       2.61    2.14                                        SD Between Run 9.71       11.94   29.10                                       CV Between Run (%)                                                                           4.99       2.98    3.21                                        SD Total       10.72      15.88   34.98                                       CV Total (%)   5.51       3.96    3.86                                        ______________________________________                                    

Two sets of calibrators and controls were prepared by adding knownquantities of dextropropoxyphene to human urine and X Systems DilutionBuffer to levels of 150, 200, 300, 400, 500, 900, 1000 and 1500 ng/ml. Acalibration was run with urine calibrators and both sets of calibratorswere assayed relative to this calibration. In the following Table 3, "%Recovery" equals 100×(measured concentration in buffer divided bymeasured concentration in urine).

                  TABLE 3                                                         ______________________________________                                        Target    Concentration                                                                             Concentration                                           Concentration                                                                           in Buffer   in Urine                                                (ng/ml)   (ng/ml)     (ng/ml)     % Recovery                                  ______________________________________                                        150       155.87      157.97       98.7                                       200       208.46      199.91      104.3                                       300       290.89      287.74      101.1                                       400       392.98      401.29       97.9                                       500       506.07      493.98      102.4                                       900       932.92      901.39      103.5                                       1000      1020.65     1003.00     101.8                                       1500      1447.40     1489.22      97.2                                       ______________________________________                                         Average Recovery = 100.9 plus or minus 2.6%                              

Various test compounds were assayed (to determine cross-reactivity) withthe dextropropoxyphene assay after a known quantity of the test compoundwas added to drug-free human urine and In the following Tables 4 and 5,"% Cross-Reactivity" equals 100× ("Concentration Found" divided by the"Concentration Added"). Cross-reactivity was tested fornordextropropoxyphene (N-Norpropoxyphene). The results fordextropropoxyphene, is summarized in Table 4 below. From the results inTable 4, it can be seen that the invention can provide immunoassayshaving advantageously high-cross reactivity for nordextropropoxyphene(NDP).

                  TABLE 4                                                         ______________________________________                                        CROSS-REACTIVITY/NORDEXTROPROPOXYPHENE                                                  Concentration                                                                             Concentration                                                                             % Cross-                                    Test Compound                                                                           Added (ng/ml)                                                                             Found (ng/ml)                                                                             Reactivity                                  ______________________________________                                        NDP       1,500       445.13      29.7                                        NDP       1,000       394.62      39.5                                        NDP       400         235.78      58.9                                        NDP       300         204.67      68.2                                        NDP       200         160.39      80.2                                        ______________________________________                                    

Cross-reactivities likewise were measured for compounds (interferants)that have similar chemical structure or are used concurrently by humans.The results are summarized in the following Table 5. In Table 5, "ND*"means "None Detected" meaning that the concentration is less than thesensitivity of the assay. From Table 5 it can be seen that the inventioncan provide immunoassays having advantageously low cross-reactivity forpotential interferants.

                  TABLE 5                                                         ______________________________________                                        CROSS-REACTIVITIES/INTERFERANTS                                                          Concentration                                                                             Concentration                                                                             % Cross-                                   Test Compound                                                                            Added (ng/ml)                                                                             Found (ng/ml)                                                                             Reactivity                                 ______________________________________                                        Amitriptylene                                                                            1,000,000   228.81      <0.1                                       "          100,000     74.75       <0.1                                       "          50,000      54.50        0.1                                       "          10,000      ND*         --                                         Brompheniramine                                                                          100,000     56.84       <0.1                                       "          50,000      40.89       <0.1                                       Chlorpheniramine                                                                         1,000,000   130.50      <0.1                                       "          100,000     ND*         --                                         Chlorpromazine                                                                           100,000     101.59       0.1                                       "          50,000      76.35        0.2                                       "          10,000      ND*         --                                         Clemastine 100,000     59.53       <0.1                                       "          50,000      ND*         --                                         Cyclizine  100,000     48.03       <0.1                                       Diphenhydramine                                                                          100,000     56.01       <0.1                                       "          50,000      ND*         --                                         Ethinyl Estradiol                                                                        100,000     46.78       <0.1                                       Fluoxetine 1,000,000   294.35      <0.1                                       "          100,000     113.22       0.1                                       "          50,000      79.83        0.2                                       "          10,000      ND*         --                                         Methadone  100,000     50.93       <0.1                                       "          50,000      ND*         --                                         Methapyrilene                                                                            100,000     44.96       <0.1                                       Promethazine                                                                             100,000     46.07       <0.1                                       "          50,000      ND*         --                                         Trihexyphenidyl                                                                          100,000     53.54       < 0.1                                      "          50,000      49.04       <0.1                                       "          10,000      ND*         --                                         Tripelennamine                                                                           1,000,000   155.73      <0.1                                       "          100,000     47.21       <0.1                                       "          50,000      ND*         --                                         ______________________________________                                    

What is claimed is:
 1. A substantially optically pure hapten, useful inan immunoassay for dextropropoxyphene and/or nordextropropoxyphene, saidhapten corresponding to the formula (IX): ##STR14## wherein Z is--NH--;X is --CHO, --COOH, --NH₂, or --COOR in which R is a C₁ -C₃ alkylgroup; m is 0 or 1; and n is an integer of from 1 to
 3. 2. The hapten ofclaim 1 wherein m=0 and n=3.
 3. The hapten of claim 2 wherein X is--CHO.
 4. A fluorescent tracer derived from a substantially opticallypure compound, said tracer being useful in an immunoassay fordextropropoxyphene and/or nordextropropoxyphene and corresponding to theformula (XI): ##STR15## wherein Z is --NH--;Y is --NH--, --(CO)NH--, or--NH(CO)--; m is 0 or 1; n is an integer of from 1 to 3; and FL is afluorescence-conferring moiety.
 5. The fluorescent tracer of claim 4wherein m=1 and n=1.
 6. The fluorescent tracer of claim 5 wherein Y is--(CO)NH--.
 7. The fluorescent tracer of claim 4 wherein saidfluorescence-conferring moiety is a monovalent residue of fluorescein ora monovalent residue of a fluorescein derivative.